Project Summary Cellular transplantation has emerged as a promising therapeutic approach for myocardial repair. However, several critical issues remain to be addressed which include, but are not limited to: 1) low donor cell engraftment rate (ranging from 0.1-10% in previous publications); 2) lack of knowledge on the mechanisms underlying the in vivo beneficiary effects of grafted cells. Understanding the in vivo effects of grafted cells may promote the development of more effective cardioprotective strategies. Several groups reported that applying prefabricated cardiac tissue, a cardiac muscle patch (CMP) made of hiPSC-derived cardiac cells, effectively increased engraftment rate. We recently established a novel strategy which has been demonstrated to significantly enhance engraftment rate. Specifically, we established a human induced pluripotent stem cell (hiPSC) line which carries a transgene encoding for the human CCND2 (Cyclin D2) driven by the cardiomyocyte specific ?-myosin heavy chain (?-MHC) promoter. CCND2-overexpressing hiPSC- derived cardiomyocytes (hiPSC-CCND2OECMs) exhibits increased cell cycle activity and cell proliferation compared with genetically nave hiPSC-CMs expressing wild-type levels of CCND2 (hiPSC-CCND2WTCMs). In a mouse model of myocardial infarction (MI), the number of engrafted cells was tripled in hearts injected with hiPSC-CCND2OECMs compared to those receiving hiPSC-CCND2WTCMs 4 weeks post MI and transplantation, resulting in significantly smaller infarct size and improved cardiac function. These data suggests that transgenic CCND2 overexpression in hiPSC-CM grafts constitutes a viable approach to enhance engraftment and restore function in ischemic heart disease. The proposal will develop a novel human cardiac muscle patch with hiPSC-CCND2OECMs (designated as hCMP- CCND2OECMs), assess their capability to continuously remuscularize the injured heart in the long term and ultimately replace the scar tissue, and test their safety and translational potential in a large animal model (the pig MI model). In the case transplanted hCMPs improve cardiac function in the chronically infarcted pig hearts, the proposal also determine if this functional improvement is attributable to remuscularization or other mechanisms. Specifically, we aimed to address the yet unanswered, but profoundly important, question whether hCMPs improve cardiac function via direct donor-host coupling in pig MI model. Our long term goal is to develop a heart regeneration strategy that can be translated to humans. Two Specific Aims are proposed. Specific Aim 1 will test the hypothesis that this novel hCMP continuously remuscularize injured myocardium, replacing transmural scar tissue, and improve cardiac function in infarcted pig hearts. We will determine (i) the impact of patch transplantation on cardiac structure and function, and (ii) safety of patch transplantation (susceptibility to inducible arrhythmias, and risk of tumor formation). Specific Aim 2 test the hypothesis that the magnitude of functional changes positively correlates with the number of donor cell-derived CMs. We will determine whether (i) donor cell survival is required for sustained improvement of cardiac function, and (ii) whether improvement in cardiac function is mediated, at least in part, by electromechanical coupling of transplanted donor myocytes.